Voltage-dependent and Calcium-dependent potassium channels are key molecular elements in the control of membrane excitability and signaling in athe nervous system. They play critical roles in the pacemaker activity of endogenously active neurons and are important in the modulation of synaptic function. Potassium channels have been shown to play a central role in the control and modulation of transmitter release in a number of systems. Alterations in potassium channels in presynaptic terminals of Shaker Drosophila mutants leads to delayed action potential repolarization and hyperexcitability at the neuromuscular junction. These effects underlie the behavioral defects in the mutant files. In Aplysia, modulation of potassium channels by neurotransmitters has been shown to strengthen synapses involved in sensitization and associative learning. A detailed understanding of the molecular mechanisms of potassium channel function will provide insights into normal and pathological synaptic function as well as intro synaptic plasticity. In addition, the advances in the knowledge of channel function should provide a better framework for designing therapeutic agents for pathological conditions involving cellular signal transduction processes. We propose to continue the study of inactivation of Shaker potassium channels. Our previous work has established a "Ball and Chain" mechanism for rapid inactivation that involves block of the internal mouth of the channel by an amino-terminal domain of the Shaker polypeptide. Progress made during the previous grant period has led to a detailed understanding of the role of specific amino acids in the amino-terminal domain in the mechanism of inactivation, and have defined the general biophysical properties of the binding site on the mouth of the channel. A major goal during the next funding period will be to define the amino-acid contributors to the inactivation "receptor site". previous work by other labs and ours has implicated three regions as potential parts of the receptor site. We will use chimeric channels and our range of N-terminal peptides to study these and other regions in more detail to get a better understanding of the properties of the receptor sites and its interaction with the N-terminal inactivation domain. With the recent cloning of large-conductance Calcium-activated potassium channels, studies of the molecular mechanisms of voltage and calcium dependent gating in these important channels are now feasible. These channels offer several advantages for such studies that make them an important tool for the study of channel gating mechanisms.. We will continue our studies of gating in these channels with a direction towards understanding the interactions between calcium and voltage-dependent gating.